Quantitative determination of risedronate in urine by spe-lc-ms-ms

ABSTRACT

The present invention is directed to a SPE-LC-MS-MS method for quantitatively determining risedronate in a urine sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/554,111, filed Sep. 4, 2009 which is a continuation of InternationalPatent Application No. PCT/US2008/055849, filed Mar. 5, 2008 whichclaims priority to U.S. Provisional Patent Application Ser. No.60/893,444, filed Mar. 7, 2007, the entire content and disclosure ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is directed to a SPE-LC-MS-MS method forquantitatively determining risedronate in a urine sample.

BACKGROUND OF THE INVENTION

Bisphosphonates inhibit bone resorption and are effective treatments formetabolic bone diseases including osteoporosis and Paget's disease (I.Elomaa, C. Blomqvist, L. Porkka, T. Holmstrom, T. Taube, C.Lamberg-Allardt, G. H. Borgstrom. Lancet 1985; i:1155; J. P. Kosonen, J.Pharm Biomed Anal. 1992; 10:881; J. A. Cantril, H. M. Buckler, D. C.Anderson, Ann. Rheumatol. Dis. 1986; 45:1012; H. Fleisch, Horm. Metab.Res. 1997; 29:145). The prevention of bone resorption results frominhibitory effects on the function of mature osteoclasts. Severalbioanalytical methods have been published for bisphosphonates. Ingeneral, those methods are mainly based on ion-exchange and ion-pairchromatography with UV, fluorescence (with a pre or post columnderivatization), conductivity, flame photometric phosphorus selective,refractive index and explorative light-scattering detection (V.Virtanen, L. H. J. Lajunen, J. Chromatogr. 1993; 617:291; V. Virtanem,L. H. J. Lajunen, Talanta 1993; 40: 661; S. E. Meek, D. J. PietrzykAnal. Chem. 1988; 60:1397; M. J. Lovdahl, D. J. Pietrzyk, J. Chromatogr.A 1999; 850:143; R. Niemi, H. Taipale, M. Ahlmark, J. Vepsalainen, T.Jarvinen, J. Chromatogr. B 1997; 701:97; T. L. Chester, Anal. Chem.1980; 52:1621).

The technique of GC-MS, combined with acylation and silylation, has beenused to determine Risedronate in human urine (D. Y. Mitchell, R. A.Eusebio, L. E. Dunlap, K. A. Pallone, J. D. Nesbitt, D. A. Russell, M.E. Clay, P. J. Bekker, Pharm. Res. 1998; 15:228). The more sensitivemethod for analysis of Risedronate in human urine has been achievedusing enzyme linked immunosorbent assay (ELISA) (D. Y. Mitchell, M. A.Heise, K. A. Pallone, J. D. Nesbilt, M. E. Clay, J. D. Nesbitt, D. A.Russell, C. W. Melson, J. Clin. Pharmacol. 1999; 48:536). Thecolumn-switching ion-pair HPLC with UV detection has also been reportedto quantify the Risedronate in human urine (P. T. Vallano, S. B.Shugars, W. F. Kline, E. J. Woolf, B. K. Matuszewski, J. Chromatogr. B1003; 794:23). Although some of these methods showed a very highsensitivity, they are all very complicated and time-consuming.

Recently, more effort has been put on the development of LC/MS/MS methodfor risedronate with post-extraction or on cartridge derivertization (L.S. Zhu, V. N. Lapko, J. W. Lee, Y. J. Basir, C. Kafonek, R. Olsen, C.Briscoe, Rapid commun Mass Spectrom. 2006, 20: 3421; S. Turcotte, J.Couture, F. Vallee, AAPS PharmSci Vol. 5, No. 4, Abstract M1357 (2003).

SUMMARY OF THE INVENTION

The present invention is directed to a method for quantitativelydetermining risedronate in a urine sample comprising:

-   -   a) adding an internal standard to the urine sample;    -   b) applying the urine sample to an Oasis HLB cartridge, wherein        the cartridge has been pre-conditioned with methanol;    -   c) washing the cartridge with about 1% (v/v) TEA in water and        about 1% (v/v) formic acid in methanol;    -   d) eluting risedronate, at least once, with a mixture of about        60% (v/v) methanol and about 40% (v/v) water containing about 3        mM EDTA under vacuum;    -   e) evaporating the eluted solution and reconstituting with a        mixture of 10% (v/v) methanol and 90% (v/v) 0.05 M NH₄Ac—NH₄OH        buffer to provide a sample mixture of risedronate and the        internal standard; and    -   f) analyzing the sample mixture with a LC-MS/MS system.

The aforesaid SPE-LC-MS-MS method for quantitative determination ofrisedronate in a urine sample is relatively simple, sensitive, preciseand accurate, and more fully discussed with the aid of the followingfigures and detailed description below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the ESI-MS Spectrum of Risedronate.

FIG. 2 is the ESI-MS Spectrum of Deoxy-risedronate.

FIG. 3 is the Chromatogram of Risedronate in Mouse Urine (LLOQ 10ng/mL), wherein the intensity of the peak at retention of 2.27 minute ismore than three times of the intensity of the same peak in the HPLCSpectrum of Blank Mouse Urine Sample. This means that the risedronateconcentration at 10 ng/mL in mouse urine can be quantified.

FIG. 4 is the Chromatogram of Mouse Control Urine.

FIG. 5 is the Typical Chromatogram of Risedronate and Deoxy-Risedronate.

FIG. 6 is the Typical Calibration Curve of Risedronate in Mouse Urine.

DETAILED DESCRIPTION OF THE INVENTION Definitions and Abbreviations

As used above, and throughout the description of the invention, thefollowing abbreviations, unless otherwise indicated, shall be understoodto have the following meanings:

-   -   amu Atom mass unit    -   Cps Count per second    -   CV % Percent coefficient of variation    -   Diff % Percentage difference between theoretical concentration        and measured concentration    -   EDTA Ethylenediaminetetraacetic acid    -   ESI Electrical Spray Ionization    -   HPLC High Pressure Liquid Chromatography    -   LC-MS High Pressure Liquid Chromatography-Mass Spectrometry    -   LLOQ Lower limit of quantity    -   MRM Multiple reaction monitoring    -   MS Mass Spectrometry    -   m/z Mass/charge    -   NH₄Ac Ammonium acetate    -   NH₄OH Ammonium hydroxide    -   SD Standard deviation    -   SPE Solid phase extraction    -   TEA Triethylamine    -   v/v Volume/volume

As used above, and throughout the description of the invention, thefollowing terms, unless otherwise indicated, shall be understood to havethe following meanings.

“Deoxy-Risedronate” means (1-phosphono-2-pyridin-3-yl-ethyl)-phosphonicacid, having the following chemical structure:

“Inertsil Phenyl-3 HPLC column” is manufactured and sold by Varian, Inc.

“Internal standard” is a chemical substance that is added in a constantamount to samples, blank and calibration standards in an analysis. Thischemical substance is used for calibration by plotting the ratio of theanalyte signal to the internal standard signal as a function of theanalyte concentration of the standards. This is done to measure andcorrect for the loss of analyte during sample preparation or sampleinlet. Particularly, the internal standard is a compound that hassimilar, but not same, chemical structure to the analyte.

“Oasis HLB cartridge” is manufactured and sold by Waters Corporation.The size of the cartridge is dependent upon the risedronateconcentration in the urine sample. The lower risedronate concentrationthe urine sample has, the bigger size of the cartridge, and the largervolume of urine sample and the solution to wash the cartridge and eluterisedronate should be used.

“Risedronate” means(1-hydroxy-1-phosphono-2-pyridin-3-yl-ethyl)-phosphonic acid, having thefollowing chemical structure:

“Urine sample” means mammalian urine sample, including human urinesample.

PARTICULAR EMBODIMENT OF THE INVENTION

One particular embodiment of the invention is the method forquantitatively determining risedronate in a urine sample, wherein theurine sample is a mouse or rat urine sample.

Another particular embodiment of the invention is the method forquantitatively determining risedronate in a urine sample, wherein theinternal standard is deoxy-risedronate.

Another particular embodiment of the invention is the method forquantitatively determining risedronate in a urine sample, wherein theOasis HLB cartridge is a 30 mg Oasis HLB catridge.

Another particular embodiment of the invention is the method forquantitatively determining risedronate in a urine sample, wherein theinternal standard is deoxy-risedronate that is added as a solution inwater; more particularly as an about 10 μg/mL solution in water.

Another particular embodiment of the invention is the method forquantitatively determining risedronate in a urine sample, wherein theamount of the urine sample is about 0.4 mL.

Another particular embodiment of the invention is the method forquantitatively determining risedronate in a urine sample, wherein thecartridge has been pre-conditioned with about 1 mL of methanol.

Another particular embodiment of the invention is the method forquantitatively determining risedronate in a urine sample, wherein thecartridge has been pre-conditioned with about 1 mL of methanol andfollowed by about 0.5 mL of 1% (v/v) TEA in water.

Another particular embodiment of the invention is the method forquantitatively determining risedronate in a urine sample, wherein thecartridge is washed with about 0.5 mL of about 1% (v/v) TEA in water andabout 0.5 mL of about 1% (v/v) formic acid in methanol.

Another particular embodiment of the invention is the method forquantitatively determining risedronate in a urine sample, wherein theamount of the mixture of about 60% (v/v) methanol and about 40% (v/v)water containing about 3 mM EDTA is about 1.5 mL.

Another particular embodiment of the invention is the method forquantitatively determining risedronate in a urine sample, whereinrisedronate is eluted twice with about 0.75 mL of the mixture of about60% (v/v) methanol and about 40% (v/v) water containing about 3 mM EDTA.

Another particular embodiment of the invention is the method forquantitatively determining risedronate in a urine sample, wherein theamount of the mixture of 10% (v/v) methanol and 90% (v/v) 0.05 MNH₄Ac—NH₄OH buffer is about 100 μL.

Another particular embodiment of the invention is the method forquantitatively determining risedronate in a urine sample, wherein theinternal standard is deoxy-risedronate, and analyzing the sample mixturewith a LC-MS/MS system comprises separating risedroante anddeoxy-risedronate by HPLC on an Inertsil Phenyl-3 HPLC column bygradient elution with a mixture of solution A and solution B, whereinthe amount of solution A is increased from about 10% (v/v) to about 95%(v/v), and wherein the solution A is about 90% (v/v) methanol in water,and solution B is about 10 mM ammonium acetate-acetic acid buffer with2% (v/v) triethylamine.

Another particular embodiment of the invention is the method forquantitatively determining risedronate in a mouse or rat urine sample,comprising:

-   -   a) adding about 100 μL of about 10 μg/mL dexoy-risedronate        solution in water to about 0.4 mL of the mouse or rat urine        sample;    -   b) applying the mouse or rat urine sample to a 30 mg Oasis HLB        cartridge, wherein the cartridge has been pre-conditioned with        about 1 mL of methanol and followed by about 0.5 mL of 1% (v/v)        TEA in water;    -   c) washing the cartridge with about 0.5 mL of 1% (v/v) TEA in        water and about 0.5 mL of 1% (v/v) formic acid in methanol;    -   d) eluting risedronate with about 1.5 mL of a mixture of about        60% (v/v) methanol and about 40% (v/v) water containing about 3        mM EDTA under vacuum;    -   e) evaporating the eluted solution and reconstituting with about        100 μL of a mixture of about 10% (v/v) of methanol and about 90%        (v/v) of about 0.05 M NH₄Ac—NH₄OH buffer to provide a sample        mixture of risedronate and dexoy-risedronate; and    -   f) analyzing the sample mixture with a LC-MS/MS system, wherein        risedronate and dexoy-risedronate are separated by HPLC on an        Inertsil Phenyl-3 HPLC column by gradient elution with a mixture        of solution A and solution B, wherein the amount of solution A        is increased from about 10% (v/v) to about 95% (v/v), and        wherein the solution A is about 90% (v/v) methanol in water, and        solution B is about 10 mM ammonium acetate-acetic acid buffer        with 2% (v/v) triethylamine.

Another particular embodiment of the invention is the method forquantitatively determining risedronate in a mouse or rat urine sample,comprising:

-   -   a) adding about 100 μL of about 10 μg/mL dexoy-risedronate        solution in water to about 0.4 mL of the mouse or rat urine        sample;    -   b) applying the mouse or rat urine sample to a 30 mg Oasis HLB        cartridge, wherein the cartridge has been pre-conditioned with        about 1 mL of methanol and followed by about 0.5 mL of 1% (v/v)        TEA in water;    -   c) washing the cartridge with about 0.5 mL of 1% (v/v) TEA in        water and about 0.5 mL of 1% (v/v) formic acid in methanol;    -   d) eluting risedronate twice with about 0.75 mL of a mixture of        about 60% (v/v) methanol and about 40% (v/v) water containing        about 3 mM EDTA under vacuum;    -   e) evaporating the eluted solution and reconstituting with about        100 μL of a mixture of about 10% (v/v) of methanol and about 90%        (v/v) of about 0.05 M NH₄Ac—NH₄OH buffer to provide a sample        mixture of risedronate and dexoy-risedronate; and    -   f) analyzing the sample mixture with a LC-MS/MS system, wherein        risedronate and dexoy-risedronate are separated by HPLC on an        Inertsil Phenyl-3 HPLC column by gradient elution with a mixture        of solution A and solution B, wherein the amount of solution A        is increased from about 10% (v/v) to about 95% (v/v), and        wherein the solution A is about 90% (v/v) methanol in water, and        solution B is about 10 mM ammonium acetate-acetic acid buffer        with 2% (v/v) triethylamine.

It is to be understood that this invention covers all appropriatecombinations of the particular embodiments referred thereto.

The present invention will appear more clearly from the followingexample that is presented as an illustration only and is not to beconsidered as limiting the invention in its scope.

EXAMPLE Step 1: Preparation of Risedronate Urine Standards in Mouse orRat Urine

A 1 mg/mL risedronate stock solution is prepared by dissolvingrisedronate in HPCL grade water. 250 μg/mL, 100 μg/mL and 50 μg/mLrisedronate standard solutions are prepared by diluting 250 μL, 100 μLand 50 μL of the 1 mg/mL risedronate stock solution to 1 mL with HPLCgrade water, respectively. Appropriate dilutions of 250 μg/mL, 100 μg/mLand 50 μg/mL risedronate standard solutions are performed to yieldrisedronate working solutions of 25 μg/mL, 10 μg/mL, 5 μg/mL, 2.5 μg/mLand 1 μg/mL. Riseronate urine standards, ranging from 10 ng/mL to 2500ng/mL, are prepared by adding 4 μL of each working solution into 0.4 mLof mouse or rat control urine (i.e., urine from mouse or rat that is notdosed with risedronate).

Step 2: Sample Preparation

A 0.4 mL urine sample collected from mouse or rat that has been dosedwith risedronate is transferred to a 10×75 mm glass culture tube. 100 μLof stock internal standard solution, i.e., 10 μg/mL deoxy-risedronatesolution in water, and 100 μL of 5% TEA in water are added to all therisedronate urine standards and the urine sample, respectively.

Sample extraction is performed with a 30 mg Oasis HLB cartridge (1 mL,manufactured and sold by Waters Corporation, Catalog No.: WAT094225).The cartridges are conditioned with 1 mL methanol and followed by 0.5 mLof 1% TEA in water. The urine sample is applied to the cartridge. Thecartridge is washed with 0.5 mL of 1% TEA in water and 0.5 mL of 1%formic acid in methanol, and then the analyte and the internal standardare eluted using two consecutive elution steps with 0.75 mL of a mixtureof 60% methanol in water containing 3 mM EDTA. During the samplesextraction, a vacuum (5-15 Psi) is used at each step to pull the liquidthrough the cartridges. The eluted solution is dried under nitrogen at37° C. for 70 minutes and then the residue is reconstituted in 100 μL of10% methanol/90% 0.05 M NH₄Ac—NH₄OH buffer (pH 9.26).

Step 3: LC-MS/MS analysis

The analysis of Risedronate is performed using API 4000 LC-MS/MS system(sold by MDS Sciex), including a Shimadzu LC-10AD pump, SCL-10A VPsystem controller, Leap Technologies HTS PAL autosampler and API 4000triple quadrupole mass-spectrometer.

Risedronate and the internal standard are separated on an InertsilPhenyl-3 HPLC column (3 μL, 50×030 mm, manufactured and sold by Varian,Inc., Catalog No.: 0408-050x030) by gradient elution with a mixture ofsolution A and solution B in 6 minutes, wherein the amount of solution Ais increased from 10% to 95%. The solution A is 90% methanol in water,and the solution B is 10 mM ammonium acetate-acetic acid buffer (pH=5.0)with 2% triethylamine. The flow rate is kept constant at 0.2 mL/min. Thesample (10 μL reconstitute) is injected onto the HPLC column directly.The mass spectrometer is programmed to monitor the transition m/z281.9→463.1 for risedronate under negative mode with a collision energyof −58 volts. The m/z 265.8→479.2 is used to measure deoxy-Risedronateinternal standard. The source temperature is set at 350° C. Ananalytical run time of 6 minutes is employed.

Results Linearity

The assay for risedronate mouse or rat urine standard is linear from 10to 2500 ng/mL with an LLOQ of 10 ng/mL. The determined accuracy at LLOQis 14.8% with a precision of 17.8% (n=3). The regression parametersobtained during the validation are reported in table 1. Correlationcoefficients of 0.98 or greater for risedronate are obtained from theday-to-day analysis. A typical calibration curve is represented in FIG.6.

TABLE 1 Regression parameters for risedronate in mouse urine RegressionParameters Intercept Slope (response/ (response at Correlation CurveNo/Date concentration) concentration 0) Coefficient Within-day #10.000260 0.00208 0.9993 #2 0.000257 0.00151 0.9961 #3 0.000232 0.003000.9895 Mean 0.000250 0.00220 0.9950 SD 0.000015 CV % 6.2 Day-to-day Day1 0.000267 0.00331 0.997 Day 2 0.000229 0.000594 0.9974 Day 3 0.0002600.00208 0.9993 Mean 0.000252 0.00199 0.9979 SD 0.000020 CV % 8.0

Specificity

A small peak, at retention time of 2.27, is found in mouse controlurine, but the peak height of LLOQ (10 ng/mL) is more than three timesof the intensity of the peak in the mouse control urine (see FIG. 3 andFIG. 4). Thus, risedronate concentration at 10 ng/mL can be quantified.

Precision

Precision is expressed as the percent coefficient of variation of theconcentrations measured at each control level over the duration of thestudy. The within-day coefficient of variation for the controls in mouseurine ranged from 2.2 to 9.6%. The day-to-day precision for the controlsat 3 concentration levels ranged from 5.2 to 10.3%. The within-day andthe day-to-day precisions for the standards ranged from 0.3 to 26%.

Accuracy

The assay accuracy is expressed as the percent difference between themean determined quality control concentration and the theoretical value.

The within-day assay accuracy for the mouse urine controls ranged from−5.3 to 4.4%. The day-to-day accuracy is determined over three analysisdays using the controls at 3 concentration levels. The day-to-dayaccuracy ranged from −4.0 to 6.0%. The accuracy from the back-calculatedconcentrations of the mouse urine standards is within ±15%.

TABLE 2 The accuracy and precision of mouse urine calibration standardsof risedronate Replicate Nominal Concentration (ng/mL) Or date 10 25 50100 250 500 1000 2500 Within-day #1 7.93 20.7 49.4 100 237 422 995 2460#2 13.3 22 42.8 91.6 238 388 868 2470 #3 9.94 25.4 50.6 97 253 476 9762500 Mean 10.39 22.7 47.6 96.2 243 429 946 2477 SD 2.71 2.43 4.20 4.268.96 44.38 68.50 20.82 CV % 26.1 10.7 8.8 4.4 3.7 10.4 7.2 0.8 Diff %3.9 −9.2 −4.8 −3.8 −2.9 −14.3 −5.4 −0.9 Day-to-day Day 1 13.8 24.7 46.993.5 237 532 1120 2610 Day 2 10.7 21.5 50.6 107 248 501 1010 2640 Day 39.94 25.4 50.6 97 253 476 976 2500 Mean 11.48 23.9 49.4 99.2 246 5031035 2583 SD 2.04 2.08 2.14 7.01 8.19 28.05 75.27 73.71 CV % 17.8 8.74.3 7.1 3.3 5.6 7.3 2.9 Diff % 14.8 −4.5 −1.3 −0.8 −1.6 0.6 3.5 3.3

TABLE 3 The accuracy and precision of mouse urine quality controls ofrisedronate Nominal Concentration (ng/mL) Date 25 250 2500 Day 1 28.2257 2900 30 290 2580 26.1 236 2870 Day 2 28.9 226 2650 27.2 208 280025.5 238 2470 Day 3 27.1 244 2650 24.1 246 2670 24.4 231 2520 23.8 2042600 20.6 259 2610 Within-day (Day 3) Mean 24.0 237 2610 SD 2.31 20.857.9 CV % 9.6 8.8 2.2 Diff % −4 −5.28 4.4 Day-to-day Mean 26.0 240 2665SD 2.69 24.1 138 CV % 10.3 10.1 5.2 Diff % 4.0 −4.0 6.6

Recovery

A recovery of risedronate in extracted mouse urine as compared toextracted mouse urine blank with neat spiked is around 51.3%

TABLE 4 Recovery of risedronate from mouse urine samples (250 ng/mL)Extracted Blank Urine Extracted Urine Sample Sample + spiked neat peakReplicates (peak area) area #1 12400 30400 #2 18000 28800 #3 16600 29800#4 18100 36000 #5 14500 30300 Mean 15920 31060 SD 2447 2833 CV % 15.49.1 Recovery (%) 51.3

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof.

1. A method for quantitatively determining risedronate in a urine samplecomprising: a) adding an internal standard solution to the urine sample;b) applying the urine sample to an Oasis HLB cartridge, wherein thecartridge has been pre-conditioned with methanol; c) washing thecartridge with about 1% (v/v) TEA in water and about 1% (v/v) formicacid in methanol; d) eluting risedronate, at least once, with a mixtureof about 60% (v/v) methanol and about 40% (v/v) water containing about 3mM EDTA under vacuum; e) evaporating the eluted solution andreconstituting with a mixture of 10% (v/v) methanol and 90% (v/v) 0.05 MNH₄Ac—NH₄OH buffer to provide a sample mixture of risedronate and theinternal standard; and f) analyzing the sample mixture with a LC-MS/MSsystem.
 2. The method according to claim 1, wherein the urine sample isa mouse or rat urine sample.
 3. The method according to claim 1, whereinthe internal standard is deoxy-risedronate.
 4. The method according toclaim 1, wherein the internal standard is deoxy-risedronate that isadded as a solution in water.
 5. The method according to claim 1,wherein the internal standard is deoxy-risedronate that is added as anabout 10 μg/mL solution in water.
 6. The method according to claim 1,wherein the Oasis HLB cartridge is a 30 mg Oasis HLB cartridge.
 7. Themethod according to claim 1, wherein the amount of the urine sample isabout 0.4 mL.
 8. The method according to claim 1, wherein the cartridgehas been pre-conditioned with about 1 mL of methanol.
 9. The methodaccording to claim 1, wherein the cartridge has been pre-conditionedwith about 1 mL of methanol and followed by about 0.5 mL of 1% (v/v) TEAin water.
 10. The method according to claim 1, wherein the cartridge iswashed with about 0.5 mL of about 1% (v/v) TEA in water and about 0.5 mLof about 1% (v/v) formic acid in methanol.
 11. The method according toclaim 1, wherein the amount of the mixture of about 60% (v/v) methanoland about 40% (v/v) water containing about 3 mM EDTA is about 1.5 mL.12. The method according to claim 1, wherein risedronate is eluted twicewith about 0.75 mL of the mixture of about 60% (v/v) methanol and about40% (v/v) water containing about 3 mM EDTA.
 13. The method according toclaim 1, wherein the amount of the mixture of 10% (v/v) methanol and 90%(v/v) 0.05 M NH₄Ac—NH₄OH buffer is about 100 μL.
 14. The methodaccording to claim 1, wherein the internal standard isdeoxy-risedronate, and analyzing the sample mixture with a LC-MS/MSsystem comprises separating risedroante and deoxy-risedronate by HPLC onan Inertsil Phenyl-3 HPLC column by gradient elution with a mixture ofsolution A and solution B, wherein the amount of solution A is increasedfrom about 10% (v/v) to about 95% (v/v), and wherein the solution A isabout 90% (v/v) methanol in water, and solution B is about 10 mMammonium acetate-acetic acid buffer with 2% (v/v) triethylamine.